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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a very common method of gel electrophoresis for separating proteins by mass. SDS-PAGE was first known as the Laemmli method, after its inventor, U.K. Laemmli. The key component of this technique happens before proteins are electrophoresed; they are denatured in a detergent called sodium dodecyl sulfate (SDS). SDS denatures proteins by binding to the hydrophobic core of the protein, and gives each protein a negative charge proportionate to its mass. SDS binds to the protein in a ratio of approximately 1.4 grams of SDS per 1.0 g of protein, but can vary from 1.1-2.2 grams of SDS per gram of protein [1]. The proteins may be further treated with reducing agents, such as dithiothreitol (DTT), to break any reformed disulfide bonds, and then alkylated with iodoacetamide to prevent further reformation of disulfide bonds.

The end result of this process is that the denatured proteins become linear, so that all proteins have similar structure and can be separated according to their molecular weight. Unlike conventional gel electrophoresis, where proteins would need to be broken into linear chunks for analysis, SDS-PAGE allows for analysis of the entire protein.

For further details on gels, staining, and electrophoretic analysis, see the "Gel Electrophoresis" section of this chapter.

In isoelectric focusing (IEF), proteins are separated by an electric current that passes through a gel containing a pH gradient. A protein's charge depends on the charge of its amino acid side chains. These charges change with pH. A protein will move through the pH gradient until its charge is zero since neutral molecules are not attracted to either the cathode or anode. The pH at which the protein is neutral is called the isoelectric point.

Immobilized pH gradients (IPGs) are used for IEF because the fixed pH gradients remain stable, even at very high voltages, for a long time. Buffering compounds are used to create the pH gradients of IPGs. IPGs are cast strips of gel with plastic backing sheets, and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. [2] Isoelectric focusing is run in the same solutions that are used to extract or solubilize the proteins.

IPG strips loaded with protein must be rehydrated in rehydration/sample buffer. Rehydration can be active or passive. To load larger proteins, a small voltage is applied, causing active rehydration. After electrophoresis is complete, the focused strips can be frozen for storage.

Native-PAGE is used to separate proteins in their native states, according to differences in their charge density. The native state of a protein is its properly folded state, not denatured or unfolded. It is important in Native-PAGE that there be no denaturants present in the gel or buffer. Otherwise, proteins will not maintain their native state. Many proteins are shown to be enzymatically active after separation by native PAGE. Thus, it is used for preparation of purified and active proteins. Native-PAGE can be carried out near neutral pH to prevent denaturation by acids and bases; it is useful to study individual conformations, as well as aggregation or self-associations. Denaturation and proteolysis is further reduced by keeping the apparatus and sample cold. [3]

The mobility of proteins depends both on their charge and size. The amino acid composition and post-translational modifications of any particular protein determines its charge. Smaller proteins or those with higher charge move farther during electrophoresis than larger proteins or those with less charge.